Natural Killer T (NKT) cells are potent, regulatory T cells that have been shown to be intimately involved in the body's response to infection and tumor progression. There is also strong evidence that NKT cells regulate the immune system in such a way as to prevent autoimmunity. Activation of NKT cells may provide new therapies for viral and bacterial infections, for cancer and for a range of autoimmune conditions, such as Type I diabetes, multiple sclerosis and lupus. NKT cells are divided into two major classes, Type I (iNKT) and Type II NKT cells. Both are activated by glycolipids which serve as ligands to an antigen presenting protein, CD1d. Glycolipid activation of iNKT cells has been extensively studied for more than a decade and a half. Much less is known about the Type II cells. What is now clear is that crucial to utilizing NKT cell stimulation therapeutically is the ability to contrl the nature of the immune response. Although a number of glycolipids that elicit a selective Th1 or Th2 response from iNKT cells have been identified, the bias often disappears in vivo, or the compounds fail to active human iNKT cells. Another need in the NKT cell arena is for the identification of potent activators of sulfatide-reactive Type II NKT cells and the development of baseline structural activity relationships in these much less explored NKT cells. In collaboration with immunologists, structural biologists and physical chemists this proposal has two major thrusts: 1) To synthesize and evaluate glycolipids with structural features designed to elicit a Th1 biased response relevant for the treatment of infectious diseases and cancer. To realize this goal three hypotheses will be tested: a) The ability of some Th1 skewing glycolipids to persist as antigen-CD1d complexes in vivo allows for the prolonged activation of Type I NKT cells, which is a significant determinant for enhanced Th1 bias; b) Modifications on the 4-position of the sugar can be used to stimulate Type I NKT cells toward a Th1 biased response relevant to humans; c) A requirement for endosomal processing as a design feature can be used to induce a more pronounced Th1 bias. Each of the hypotheses is based on solid preliminary studies involving human cell lines and or a humanized mouse model. Additional evaluations, including crystallographic studies of these and related analogs, provide an opportunity to further explore the basis of Th1 bias. The second major thrust is: 2) To understand sulfatide-glycolipid structural influences on functional outcomes with sulfatide-reactive Type II NKT cells. These have received far less attention than the iNKT cells, but evidence is emerging that the activation of these Type II cells can counter- regulate the Type I NKT cells. However, there are no potent agonists for Type II cells. We propose to utilize what is known about the nature of how ligands interact with CD1d and what is emerging about Type II ligands to develop pharmacophoric models to design potent agonists for Type II NKT cells.